Chloroform Inactivation of Reovirus Hemagglutinins

The hemagglutinin of all three types of reovirus was inactivated by chloroform with little or no diminution in virus infectivity. Albumin and other proteins protected the hemagglutinin from inactivation by chloroform. When crude reovirus suspensions were centrifuged in a sucrose density gradient, two hemagglutinin fractions were collected. The fraction which sedimented more rapidly contained the bulk of infectious virus, but both lost hemagglutinin activity when extracted with chloroform. Ether extraction of the fractions did not reduce either the infectivity or hemagglutinin titre.     

Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes

Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.     

Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.     

Origin of the Vaccinia Virus Hemagglutinin

The relationship between the vaccinia virus hemagglutinin and hemadsorption was examined. Hemagglutinin synthesis was temporally related to the appearance of the hemadsorption reaction. Only chicken erythrocytes, which reacted with hemagglutinin, hemadsorbed to infected cells, and both of these reactions were inhibited by Ca(2+). The distribution of the vaccinia hemagglutinin and 5'-adenosine monophosphatase, a plasma membrane marker enzyme, in sucrose gradients was similar. Plasma membrane ghosts derived from infected cells hemadsorbed erythrocytes and yielded hemagglutinin upon sonic disruption. These data suggest that the majority of vaccinia hemagglutinin is derived from the plasma-membrane of the infected cell.     

Membrane fusion by single influenza hemagglutinin trimers. Kinetic evidence from image analysis of hemagglutinin-reconstituted vesicles

Influenza hemagglutinin, the receptor-binding and membrane fusion protein of the virus, is a prototypic model for studies of biological membrane fusion in general. To elucidate the minimum number of hemagglutinin trimers needed for fusion, the kinetics of fusion induced by reconstituted vesicles of hemagglutinin was studied by using single-vesicle image analysis. The surface density of hemagglutinin fusion-activity sites on the vesicles was varied, while keeping the surface density of receptor-binding activity sites constant, by co-reconstitution of the fusogenic form of hemagglutinin, HA(1,2), and the non-fusogenic form, HA(0), at various HA(1,2):(HA(1,2) + HA(0)) ratios. The rate of fusion between the hemagglutinin vesicles containing a fluorescent lipid probe, octadecylrhodamine B, and red blood cell ghost membranes was estimated from the time distribution of fusion events of single vesicles observed by fluorescence microscopy. The best fit of a log-log plot of fusion rate versus the surface density of HA(1,2) exhibited a slope of 0.85, strongly supporting the hypothesis that single hemagglutinin trimers are sufficient for fusion. When only HA(1,2) (without HA(0)) was reconstituted on vesicles, the dependence of fusion rate on the surface density of HA(1,2) was distinct from that for the HA(1,2)-HA(0) co-reconstitution. The latter result suggested interference with fusion activity by hemagglutinin-receptor binding, without having to assume a fusion mechanism involving multiple hemagglutinin trimers.     

Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein.

The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.     

Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids

The low pH-dependent fusion of lipid membranes induced by two types of the fatty acylated influenza viral hemagglutinin has been studied by use of an energy transfer assay. When protein bound fatty acids were released from the hemagglutinin by hydroxylamine treatment viral fusion activity was inhibited. The extent of fusion inhibition correlates with the amount of fatty acids cleaved from the hemagglutinin. Virosomes prepared from fowl plague virus containing fatty acid free hemagglutinin showed a much lower fusion activity than control virosomes containing fatty acylated hemagglutinin. The hydroxylamine treatment applied has no detectable effects on the virus other than fatty acid release from its spike glycoproteins. These results support our previous hypothesis that protein bound fatty acids are involved in the induction of membrane fusion by the influenza hemagglutinin.     

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.     

Some Properties and Characterization of Rice Seed Hemagglutinin

The phytohemagglutinin of rice seed has been purified by a sequence of steps involving fractionation with ammonium sulfate and successive chromatography on DEAE-and eMcellulose and finally gel filtration on Bio-Gel P-100. The purified rice seed hemagglutinin was shown to be homogeneous by electrophoresis on polyacrylamide gel and its molecular weight was 10,000, calculated from both the Ve/Vo value of gel filtration on Bio-Gel P-100 and the sum of the individual constituents (amino acids, sugars and metals). In addition to amino acid, the rice seed hemagglutinin contained 26.8% covalently bound carbohydrate which was identified and quantitated by gas chromatography of the acetylated alditols. Glucose was the predominant sugar with lesser amounts of glucosamine, xylose, and mannose also being present. And the rice seed hemagglutinin contained 1 g atom of calcium per molecule. The molecular weight of the rice seed hemagglutinin is smallest compared with some of phytohemagglutinins isolated from leguminous seeds and other plant sources. The rice seed hemagglutinin has the blastogenetic activity for human peripheral lymphocytes as well as Phaseolus vulgaris phytohemagglutinins or concanavalin A, jack bean hemagglutinin.     

Stereochemical structure recognized by the L-fucose-specific hemagglutinin produced by Streptomyces sp

A hemagglutinin has been purified 4000-fold from the culture filtrate of a strain of Streptomyces by affinity chromatography. The purified preparation was judged to be homogeneous by gel electrophoresis and its molecular weight was estimated to be about 70 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It may exhibit its full hemagglutinating activity in the monomer form. This hemagglutinin strongly agglutinated human blood group O erythrocytes and was inhibited by L-fucose. It was, however, distinct from the known L-fucose-specific hemagglutinins; first, the hemagglutinating activity of the purified preparation was more than 100-times stronger than that of others; second, D-mannose was a potent inhibitor of this hemagglutinin besides L-fucose but not or scarcely inhibitory to others; and third, p-nitrophenyl-beta-L-fucoside was more inhibitory to this hemagglutinin than p-nitrophenyl-alpha-L-fucoside as opposed to the case of others.     

Polar appearance and nonligand induced spreading of measles virus hemagglutinin at the surface of chronically infected cells

Fixation with glutaraldehyde (GA) and paraformaldehyde (PFA) preserved measles virus hemagglutinin at the surface of chronically infected cells. Cells fixed with PFA but not with GA exhibited hemadsorption with green monkey cells. PFA fixation, in contrast to GA fixation, also preserved the immunogenicity of measles virus hemolysin.These fixatives and the removal of the measles virus hemagglutinin from the cell surface by trypsin enabled studies of the appearance of the hemagglutinin at the surface membrane. Results obtained by immunofluorescence technique and by hemadsorption indicated that measles virus hemagglutinin appeared polarly at the cell membrane and then spread around the surface. This was substantiated by measurements of the immunofluorescence intensity at the single cell level per membrane unit and per cell, and by measuring the binding of iodinated immunoglobulins per 10⁶ cells. The appearance was inhibited by sodium azide and cytochalasin B. The spreading was not inhibited by sodium azide, but was influenced by cytochalasin B. The spreading did not proceed at 4°C. On the basis of these findings, a hypothetical model for appearance and spreading of measles virus hemagglutinin was proposed.     

Isolation, purification, and partial characterization of type V-A hemagglutinin from Escherichia coli GV-12, O1:H-

A hemagglutinin which specifically agglutinates human type A erythrocytes (mannose resistant) was isolated from the growth medium of cultures of Escherichia coli GV-12, serotype O1:H-, and purified by chromatography on Bio-Gel A-1.5 and DEAE-Sephadex A-25. The purity of the hemagglutinin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. N-terminus analysis indicated that only asparagine resides on the amino terminus. The native hemagglutinin is an aggregate exhibiting a sedimentation coefficient of 9.25, which corresponds to a molecular weight of approximately 200,000. The monomeric molecular weight was found to be approximately 16,300. Amino acid analysis indicated that the hemagglutinin consists of 131 residues, corresponding to a molecular weight of 13,400.     

Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation

The transport kinetics of the influenza virus hemagglutinin from its site of synthesis to the apical plasma membrane of Madin-Darby canine kidney cells, a polarized epithelial cell line, were studied by a sensitive tryptic assay. Hemagglutinin acquired terminal sugars, as judged by sensitivity to endo-β-N-acetylglucosaminidase H, 10–15 min after synthesis, and first appeared on the apical domain 15 min later. None of the pulse-labeled hemagglutinin accumulated on the basolateral domain. At 20°C, terminal glycosylation continued, but no hemagglutinin was detected on the cell surface within 2 hr. If the incubation temperature was raised from 20°C to 37°C, hemagglutinin was quickly externalized, demonstrating that the inhibition at low temperature was reversible.     

Lysolipids do not inhibit influenza virus fusion by interaction with hemagglutinin

The interaction of a spin-labeled lysophosphatidylcholine analog with intact and bromelain-treated influenza viruses as well as with the bromelain-solubilized hemagglutinin ectodomain has been studied. The inhibition of fusion of influenza viruses with erythrocytes by the lysophosphatidylcholine analog was similar to that observed for non-labeled lysophosphatidylcholine. Only a weak interaction of the lysophosphatidylcholine analog with the hemagglutinin ectodomain was observed even upon triggering the conformational change of the ectodomain at a low pH. A significant interaction of spin-labeled lysophosphatidylcholine with the hemagglutinin ectodomain of intact viruses was observed neither at neutral nor at low pH, whereas a strong interaction of the lipid analog with the viral lipid bilayer was evident. We suggest that the high number of lipid binding sites of the virus bilayer and their affinity compete efficiently with binding sites of the hemagglutinin ectodomain. We conclude that the inhibition of influenza virus fusion by lysolipids is not mediated by binding to the hemagglutinin ectodomain, preventing its interaction with the target membrane. The results unambiguously argue for an inhibition mechanism based on the action of lysolipid inserted into the lipid bilayer.     

Ammonium chloride slows transport of the influenza virus hemagglutinin but does not cause mis-sorting in a polarized epithelial cell line

The effects of the weak base ammonium chloride on the intracellular transport and sorting of the influenza hemagglutinin to the apical plasma membrane of polarized epithelial cells were examined in infected Madin-Darby canine kidney cells. Ammonium chloride was found to significantly retard cell surface appearance of the hemagglutinin but to have no effect on either the initial sorting or steady-state levels of hemagglutinin on the apical domain. Based on the rate of acquisition of resistance to endo H, the timed addition of ammonium chloride, and dissociation by reduced temperature incubation of cell surface appearance of the hemagglutinin from early stages of transport and processing, it was determined that the likely site of ammonium chloride action was the trans Golgi.     

Isolation and characterization of Clostridium perfringens mutants altered in both hemagglutinin and sialidase production.

The relationship between the production of hemagglutinin and sialidase activities by Clostridium perfringens was investigated by screening for mutants producing reduced levels of hemagglutinin activity. Twelve mutants were isolated; all produced reduced levels of sialidase activity and several had other altered phenotypic markers. Revertants that regained the ability to produce active hemagglutinin were isolated. All of these revertants produced increased sialidase activity. These results show that the production of hemagglutinin activity is directly related to the production of sialidase activity. Evidence is also presented that the processes of sporulation and the production of extracellular proteins are interrelated.     

Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane.     

Hemagglutinin in Cell Walls of Chlamydia psittaci

Intact purified elementary bodies (EB) of Chlamydia psittaci agglutinate chicken erythrocytes in low titer, whereas homogenates of EB and of EB cell walls agglutinate at much higher titers depending on the extent of disruption by shaking and sonication. The hemagglutinin is contained in the cell envelope and can be purified with cell wall fractions. Treatment of cell wall with sodium dodecyl sulfate completely inactivated the hemagglutinin. Purified hemagglutinin was found to have an identical polypeptide composition to EB cell walls. Preparations of purified reticulate forms, the reproductive intracellular form of the organism, were almost totally devoid of hemagglutinin.     

Purification and characterization of a hemagglutinin from Arachis hypogeae.

A. hypogaea hemagglutinin was purified by ammonium sulfate fractionation and Sepharose 6 B column chromatography. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and polyacrylamide gel electrophoresis. It has a molecular weight of 106.500 and is a tetramer of a subunit with a molecular weight of 27.000. The purified hemagglutinin agglutinated neuraminidase-treated human erythrocytes regardless of their ABO group type, but did not agglutinate intact erythrocytes. In hapten inhibition assays with simple sugars, the so-called M?kel?'s group 2 sugars, which bear the same configuration of hydroxy groups at C-3 and C-4 as D-galactopyranose, were inhibitors for this hemagglutinin. It does not contain any carbohydrate, in contrast to most phytohemagglutinins except concanavalin A and wheat germ agglutinin.     

Cloning and expression of the hemagglutinin gene of influenza virus subtype H1 in Escherichia coli

The full-length copy of the hemagglutinin gene of influenza virus was inserted into M13 phage DNA. The DNA sequence coding for the hydrophobic prepeptide was removed from the gene by oligonucleotide-directed mutagenesis. The possibilities of expression of the full-length and mutant genes in E. coli were investigated. The beta-galactosidase-hemagglutinin fusion proteins were isolated. The fusion proteins exhibited specific binding to antiviral antibodies. This binding could be competitively inhibited by excess of viral hemagglutinin, demonstrating that these fusion proteins contained antigenic determinants of hemagglutinin.